JP2016500130A - C6-C18-acylated derivative of hyaluronic acid and process for its preparation, nanomicelle composition based on it and process for its preparation, process for preparing stabilized nanomicelle composition and use thereof - Google Patents

Abstract

The present invention relates to a process for the preparation of hydrophobized hyaluronic acid (formula I) and further a method for encapsulating a biologically active substance in nano micelles of hydrophobized hyaluronan which acts as a carrier for the biologically active hydrophobic substance. About. Hyaluronan is hydrophobized through esterification of hyaluronan with long-chain carboxylic acids, which are activated by halide derivatives of 2,4,6-trichlorobenzoic acid or other organic chlorides. The In an aqueous environment, water-soluble and hydrophobized derivatives can form nanomicelles, in which nonpolar substances can be bound by noncovalent physical interactions. Nanomicelle cores are formed by hydrophobic acyl functional groups, while nanomicelle shells are formed by hyaluronic acid. Encapsulation of substances in nanomicelles can be performed by solvent exchange or sonication. Hyaluronic acid nano micelles promote the penetration of the bound substance into the local application site and allow the bound substance to migrate to individual cells. Nano micelles obtained from hydrophobized hyaluronan derivatives are useful for cosmetic and medical applications.

Description

The present invention relates to a method for the preparation of hydrophobized hyaluronic acid carrying a biologically active hydrophobic substance and the use thereof, wherein the biologically active substance is encapsulated in nano micelles of hydrophobized hyaluronan. . Hyaluronan is hydrophobized via esterification of hyaluronan with a long chain carboxylic acid, which is a halide derivative of 2,4,6-trichlorobenzoic acid or another organic chloride of R ₃ -CO-Cl. It is activated by the object. In a suitable aqueous environment, the water-soluble hydrophobized derivatives form nanomicelles in which nonpolar substances can be bound by noncovalent physical interactions. Nanomicelle cores are formed by hydrophobic acyl functional groups, while nanomicelle shells are formed by hyaluronic acid. Encapsulation of substances in nanomicelles can be performed by solvent exchange or sonication. Hyaluronic nanomicelles support the penetration of bound substances in topical applications and allow the bound substances to migrate into individual cells. The invention further relates to a method of preparing a stabilized nanomicelle composition. Nano micelles obtained from hydrophobized hyaluronan derivatives are useful for cosmetic and pharmaceutical applications.

Hyaluronic acid is an important polysaccharide consisting of two repeating units β- (1,3) -D-glucuronic acid and β- (1,4) -N-acetyl-D-glucosamine. It is characterized by a high molecular weight in the range of 5 × 10 ^{4 to} 5 × 10 ⁶ depending on the method of isolation and the initial materials used. Hyaluronic acid, especially its sodium salt known as hyaluronan, is an essential component of connective tissue and joint fluid. Furthermore, it plays an important role in many biological processes such as hydration, organization of proteoglycans, cell differentiation, proliferation and angiogenesis. Polysaccharides with strong hydrophilicity are water soluble in salt form within the entire pH range.

Hyaluronic acid-based carrier system Hyaluronic acid cannot act as an effective carrier for hydrophobic substances because it is hydrophilic in its natural form. For this reason, hydrophobic functional groups must be attached to the polymer chain of hyaluronic acid. If the amount and length of such a hydrophobic functional group is sufficient, a self-aggregation process involving that functional group is initiated, so that a hydrophobic domain can be formed within the structure of hyaluronan. Subsequently, small molecules of water-insoluble substances can bind to such domains by non-covalent bonds. In the literature, the resulting structure is often called polymer nanomicelle, and the micelle core is hydrophobic, so small non-polar molecules are dissolved, while the shell is hydrophilic, so the polymer micelle is aqueous. It becomes possible to dissolve in the environment. Polymer micelles with dimensions (diameter) of less than 200 nm are called nano micelles.

  Support systems formed by polymer micelles based on modified hyaluronic acid are known for complexes of hyaluronan with alkylamines (Liu et al., 2011) and formic acid. However, the presence of formamide, which is highly toxic and teratogenic, is considered essential for the preparation of the above hyaluronan derivatives. In order to obtain redox-sensitive micelles, a similar polymer micelle preparation method was employed (Li et al., 2011). It is clear that such micellar systems cannot be used for biological applications due to the presence of highly toxic reagents.

Conjugation of hyaluronic acid with other polymers (polymers of lactic acid or glycolic acid) via a bond mediated by the carboxyl group of D-glucuronic acid and possibly through the incorporation of low molecular weight substances is described in US Pat. , 767,806, where the authors describe the biocompatibility of the polymer, but have not been explained or proven by testing. In another case, the low molecular weight hyaluronan (9-45 kDa) is covalently modified (at the carboxyl group position of glucuronic acid) by various chain lengths of hydrophobic amines and positively charged spermines used as cationic segments. (Shen, Li, Tu and Zhu, 2009). The latter purpose is to prepare a carrier for the gene. However, acute and subacute toxicity limits the use of spermine for these purposes (Til, Falke, Prinsen and Willems, 1997). The critical micelle concentration of the formed polymer micelle having a diameter of 125 to 555 nm was measured to be 0.04 mg · mL ⁻¹ or more. In addition to the critical micelle concentration value becoming excessively high and thus the micelle system not being diluted to the maximum (eg in the bloodstream), micelles of the above dimensions are suitable candidates for passive dispersion of drugs in the human body. I don't think so. This is because the size of the polymer micelle is responsible for its ability to develop an infarcted lesion due to the tumor site or vein wall rupture. In such cases, polymer micelles with a diameter of 20-100 nm are preferred (Wang, Mongayt and Torchilin, 2005).

  Modification of the carboxyl group of glucuronic acid was used to prepare polymer hyaluronan micelles based on electrostatic interaction between negatively charged hyaluronic acid and positively charged styrylpyridinium (Tao, Xu, Chen, Bai and Liu, 2012) ). However, such interactions play an important role in the regulation of neurotransmitter release from neurons, but the interaction of styrylpyridinium with nerve endings and muscarinic receptors has been well elucidated so far. The in vivo use of such polymeric micelles is limited (Mazzone, Mori, Burman, Palovich, Belmonte and Canning, 2006).

Patent documents US 7,993,678 and WO 2007/033677 claim a process for the preparation of alkyl / aryl-succinic acid derivatives of hyaluronan, which derivatives can also be used for the encapsulation of active non-polar substances. In the latter case, the modification includes the primary hydroxyl group of hyaluronan, while the carboxyl group remains unchanged. The disadvantage of the above modification reaction is the alkaline pH range (pH 8.5 to 9.0) where the reaction between the cyclic anhydride and hyaluronan occurs. In fact, such an alkaline pH value initiates the hydrolysis of the anhydride, which reduces the efficiency of the modification process. This is particularly noticeable on an industrial scale. In the alkyl / aryl-succinic acid derivative of hyaluronan prepared by the above method and having a substitution degree of 44%, the ability to form (aggregate) micelles in an aqueous environment has an individual concentration of 0.003 to 0.004 mg · Clarified when greater than mL ^-1 . The dimensions observed with polymer micelles ranged from 50 to 200 nm. However, disadvantages of such derivatives, modified alkyl / aryl - is the increase in overall negative charge of hyaluronan due to the presence of groups ^- in succinic acid functional groups additionally COO. The negative charge of the molecule can have a rather detrimental effect on the interaction between the cell and the individual carrier system (Wang, Mongayt and Torchilin, 2005). One of the limiting factors when using derivatives prepared by the above method for injection is its low solubility (Eenschooten, Guillaumie, Kontogeorgis, Stenby and Schwach-Abdellaoui, 2010). Another disadvantage of such derivatives is the instability of ester bonds during heat sterilization processes such as autoclaving. Patent documents US 7,993,678 and WO 2007/033677 only describe a method for directly dissolving a non-polar substance in an alkyl / aryl-succinic acid derivative solution of hyaluronan, such as a stable emulsion form. The main disadvantage of the direct method of encapsulating non-polar substances is the low binding capacity of the resulting polymer micelles (Kedar, Phutane, Shidhaye and Kadam, 2010). The above patent claims the use of a modified hyaluronan structure for a carrier system, but does not provide a further method with the potential to attach a hydrophobic substance to a given hyaluronan structure other than an emulsion system. . For this reason, there is a complete lack of providing a polymer support system with sufficient binding capacity, which is one of the fundamental properties required for realistic applicability evaluation. Furthermore, no details on cytotoxicity and cell interactions are given, so we conclude whether the claimed structure is actually applicable to the active transfer of hydrophobic substances into cells This is impossible and this conclusion is essential for medical applications.

  Another publication (Smejkalova, Hermannova, Sulakova, Prusova, Kucerik and Velebny, 2012) describes the hydrophobic domain of hyaluronan, from the aggregation of covalently linked C6-acyl chains to hyaluronan. It will occur. However, it cannot be said that the derivatives described in this publication are completely free of residual solvents. Also, the publication does not mention the formation or characterization of polymer micelles. Furthermore, the symmetric anhydrides described in the above publications cannot be used to form bonds between long alkyl chains. Carboxylic acids with long aliphatic chains are very expensive and at least one mole of acid is lost during the preparation of one mole of final reagent. The publication does not mention the cytotoxicity of the prepared derivatives.

  The preparation of the polysaccharide ethyl butyrate containing an equivalent pharmaceutical composition is claimed in patent EP0941253. However, the claimed preparation method allows only a very low degree of substitution (up to 3%). The amount of hydrophobic material bound to the derivative prepared by non-covalent bonding is adversely affected by such a low degree of substitution. Ethyl butyrate hyaluronan was further prepared according to patent WO2005 / 092929 except under non-aqueous conditions. Therefore, conversion of hyaluronan to quaternary ammonium salts may be accompanied by degradation of hyaluronan. The degree of substitution obtained is less than 0.1% and thus such ester derivatives are not suitable for the preparation of carrier systems. Similar results were obtained after simultaneous esterification of hyaluronan with butyric anhydride and retinoic acid chloride (WO2004 / 056877).

Modified hyaluronan (HA)-[O (C = O) NH-M] _P (wherein M represents a modified unit comprising an alkyl functional group C _2-16 and p represents a multiple of 3 to 4) and pharmaceutical Micelle compositions based on chemically active molecules are claimed by patents US2010 / 0316682 and EP1538166A1. The main disadvantage of such derivatives is dibutyltin laurate, which is known as a substance with immunotoxicity and teratogenic potential and is used in the modification of hyaluronan. This material is often used in modification methods related to the manufacture of adhesives and is recorded in the European Environmental Organization because of its acute toxicity (Boyer, 1989). Another disadvantage of the claimed derivative is that it is conjugated to a polymer, such as polyethylene glycol, which is heterogeneous to the human body, and when used for intravenous or topical administration, an inflammatory response Cause cytotoxic degradation products. In addition, when polymer micelles are repeatedly applied, polyethylene glycol forms hydrophilic segments and the formation of anti-PEGic IgM antibodies accelerates the elimination of these micelles from the bloodstream (Gong, Chen, Zheng, Wang and Wang ,year 2012).

Paclitaxel was successfully incorporated into hyaluronan micelles modified by lactic acid-glycolic acid copolymer (PLGA) (Kim, Lee, Jang and Park, 2009). This uptake was performed using a dialysis method in which both the polymer and the individual binding substances were dissolved in DMSO and the resulting solution was dialyzed against H ₂ O. In this case, the binding capacity of the prepared polymer micelle was 4.5% by weight. The main disadvantage of such a carrier system is the presence of PLGA polymer, which is foreign to the human body and not a complete biodegradation system. Another disadvantage is that DMSO remains in the final product.

Modification of hyaluronan with long-chain carboxylic acids Modification of polysaccharides with carboxylic acids in most cases requires the anhydride of a given commercially available acid (WO1996 / 035720, WO2007 / 033677, (Smejkalova, Hermannova, Sulakova , Prusova, Kucerik and Velebny, 2012), EP0893451). The main disadvantage of such commercial anhydrides is the sensitivity to hydrolysis and the possible presence of impurities. In addition, several acid anhydrides (eg, undecane-carboxylic acid) are not commercially available. Some of the available anhydrides are very expensive (eg, oleic, linoleic or linolenic anhydrides). Therefore, the large scale preparation of modified polysaccharides becomes difficult due to the availability, high price and instability of such anhydrides.

  The acid anhydride can be replaced with other acid derivatives that can be used for esterification of hyaluronan. Patent WO2010 / 105582 claims a method for the activation of carboxylic acids with ethyl chloroformate in non-aqueous conditions, where O-acyl-O'-alkyl carbonates are formed, which is then esterified with hyaluronan. Can be used for The disadvantage of such activation is that it produces toxic and potentially explosive gases. Similar activation methods with ethyl chloroformate are disclosed in patents U.S. 3,720,662 and CZ20060605.

  Another known method is based on the esterification of polysaccharides with carboxylic acids in the presence of imidazole (U.S. 2012/0172587). However, the claimed preparation method requires a high reaction temperature (90 to 200 ° C.) that cannot be applied to hyaluronan because hyaluronan is decomposed at high temperatures.

  European patent EP0893451 claims the esterification of polysaccharides with carboxylic anhydrides using a supercritical extraction method. The disadvantages of this esterification procedure are the high pressure required and the high equipment costs.

  For this reason, it is very important to find an alternative to long chain carboxylic acid activation as a method available in situ. One possible technical solution is based on the activation of carboxylic acids by derivatives of 2,4,6-trichlorobenzoic acid with the formation of anhydrides. For the first time, 2,4,6-trichlorobenzoic anhydride was used in combination with DMAP catalyst to rapidly esterify macrocycles under mild reaction conditions (Inanaga, Hirata, Saeki, Katsuki and Yamaguchi, 1979). Year). However, since the exothermic reaction may involve degradation of individual polysaccharides, this esterification method has not yet been applied to the modification of hyaluronic acid polysaccharides.

SUMMARY OF THE INVENTION The object of the present invention is to synthesize a hydrophobized derivative of hyaluronic acid and to utilize said derivative in the form of a polymer carrier for a biologically active hydrophobic substance in an aqueous environment, Here the innate nature and biological activity of the bound substance must remain unchanged.

  Hydrophobization of hyaluronan (C6-C18) with long chain aliphatic esters is based on the direct activation of long chain carboxylic acids with the formation of 2,4,6-trichlorobenzoic anhydride (Scheme 1) . In the next step, the resulting anhydride reacts with hyaluronic acid to form an acylated derivative of hyaluronic acid. The main advantage of the activation is that the aliphatic carboxylic acid can be used directly. Unlike similar hyaluronan hydrophobization reactions that constitute the prior art, the modification of the present invention does not require the use of commercially available anhydrides. Another advantage of the activation of the present invention is that the individual reactions occur under mild conditions (room temperature to 50 ° C.) and the required time is short. Furthermore, unlike the method disclosed in patent application WO2010 / 105582, activation of the present application does not require large amounts of fatty acids or special anhydrous conditions. The activation reagent is stable and, unlike ethyl chloroformate, does not produce toxic and explosive gases when used in the reaction. Another advantage of the activating reagent of the present invention is that the reagent does not cause a reaction by a by-product and does not induce a crosslinking reaction.

スキーム１ Activation of the carboxylic acid with a derivative of 2,4,6-trichlorobenzoic acid is depicted in Scheme 1 below, along with subsequent use of the resulting activated product for hyaluronic esterification.

Scheme 1

  Furthermore, the present invention relates to the use of hydrophobized hyaluronic acid to prepare nanomicelle systems and the use of substances bound in nanomicelles of hyaluronic acid to treat skin, hair and mucous membranes, as well as other possible topical applications. It is about application. The encapsulated biologically active substance is bound in hyaluronan nanomicelles by non-covalent physical interaction with the hydrophobic functional groups of the polymer carrier. When encapsulated in the manner described above, such substances effectively penetrate the surface skin layer (epidermis), the entire structure of the hair, and the mucosal epithelium. One advantage is that the depth of penetration of the active substance bound in the nanomicelle is greater than the depth of the substance when dissolved directly in a non-polar solvent.

  Compared to similar polymer systems (such as those disclosed in patents US7,993,678 and WO2007 / 033677), hyaluronan nanomicelle conjugates unexpectedly show a critical micelle concentration 3 to 10 times lower while at the same time salinity environment And relatively high stability in an aqueous environment. Such very low critical micelle concentration values are particularly advantageous in that intravenous administration of hyaluronan micelles is possible. Unlike most other known polymer-based systems described with reference to the prior art, the desired encapsulation can be maintained without adding lyophilization inhibitors to the solution prior to freezing Therefore, the high stability of hyaluronan micelles may be advantageous in terms of lyophilizing various materials.

  Another advantage of the hydrophobized derivative of the claimed hyaluronan is a synthetic polymer (PLGA), which is often necessary for nanomicelle formation in other cases and can induce antibody formation upon repeated administration. , PEG, etc.) and copolymers are unnecessary (Gong, Chen, Wang & Wang).

  Unlike similar polymer micelle systems, the formation of nanomicelles is not based on modified hyaluronan with an increased total negative charge, and thus the interaction between the carrier system and the cells is unaffected by such charges. .

  Some nanomicelle structures, particularly those containing longer acyl chains attached to hyaluronan, can form a gel phase in an aqueous environment. Such gel phases can be advantageously used in certain applications that require increased viscosity of the individual carrier system.

  The size of the prepared nano micelle system is mostly 20 to 100 nm, which is the optimal size for medical use, and the advantages of high permeability and retention effect (EPR effect) are obtained. In the polymer micelles described with reference to the prior art, such dimensions are not always obtained.

  Another advantage of hyaluronan micelles is related to the transfer of bound material from the hydrophobic domain of nanomicelles to the cell.

  This core encapsulation method involves the preparation of a mixture of hyaluronan dissolved in water and a biologically active substance dissolved in an organic solvent, followed by the energy decay of the hyaluronan hydration envelope and subsequent removal of the solvent from the solution. based on. In contrast to common encapsulation procedures, the above method is not based on the solubility of the polymer in organic solvents, and thus, especially important carboxyl groups that allow hyaluronan to be recognized by cell receptors. In this position, it is not necessary that the innate properties of hyaluronic acid be suppressed and that the polymer chain be significantly modified. The organic solvent preferably has a lower boiling point than water. The binding capacity of hyaluronan nanomicelles is significantly increased by complete evaporation of the residual aqueous phase and subsequent rehydration of the nanomicelles. Unbound biologically active material can be removed by a filtration step, and the resulting nanomicelles can be directly lyophilized and stored dry until the time for subsequent rehydration.

式中，ＲはＨ^＋又はＮａ^＋であり，Ｒ^１はＨ又はＣ（＝Ｏ）Ｃ_ｘＨ_ｙ又はＣ（＝Ｏ）ＣＨ＝ＣＨ‐ｈｅｔであり，ｘは５〜１７の整数であり，ｙは１１〜３５の整数であり，Ｃ_ｘＨ_ｙは直鎖又は分岐の飽和又は不飽和Ｃ_５‐Ｃ_１７鎖であり，ｈｅｔはその内容をＮ，Ｓ又はＯ原子の中から選択できる複素環又は複素芳香族基であり，少なくとも１つの繰り返し単位は１つ以上のＲ^１，‐Ｃ（＝Ｏ）Ｃ_ｘＨ_ｙ又はＣ（＝Ｏ）ＣＨ＝ＣＨ‐ｈｅｔ基を含み，ｎは１２〜４０００の範囲であるものとする誘導体に関する。好適な実施態様では，前記Ｃ_６‐Ｃ_１８‐アシル化誘導体は，式（I）においてＲはＨ^＋又はＮａ^＋であり，Ｒ^１は‐Ｃ（＝Ｏ）（ＣＨ_２）_７‐ＣＨ＝ＣＨ‐（ＣＨ_２）_７‐ＣＨ_３であるオレイル誘導体である。 The invention particularly relates to C ₆ -C ₁₈ -acylated derivatives of hyaluronic acid according to general formula (I):

In the formula, R is H ⁺ or Na ⁺ , R ¹ is H or C (═O) C _x H _y or C (═O) CH═CH-het, and x is an integer of 5 to 17. , Y is an integer from 11 to 35, C _x H _y is a linear or branched saturated or unsaturated C ₅ -C ₁₇ chain, and het can select its content from N, S or O atoms A heterocyclic or heteroaromatic group, wherein at least one repeating unit comprises one or more R ¹ , —C (═O) C _x H _y or C (═O) CH═CH—het groups, and n is It relates to derivatives that are in the range of 12 to 4000. In a preferred embodiment, said C ₆ -C ₁₈ -acylated derivative is a compound of formula (I) wherein R is H ⁺ or Na ⁺ and R ¹ is —C (═O) (CH ₂ ) ₇ —CH═. It is an oleyl derivative that is CH— (CH ₂ ) ₇ —CH ₃ .

Furthermore, the present invention relates to a method for preparing the above-mentioned hyaluronic acid derivative. In the presence of a base and a catalyst in a mixed solution of water and a water-miscible aprotic solvent, hyaluronic acid is 2,4,6-trichloro. Reacts with C ₆ -C ₁₈ -carboxylic acid activated by chlorides of benzoic acid or organochlorine compounds of R ₃ -CO-Cl, R ₃ may optionally be heteroaromatic or aliphatic containing an aromatic functional group Or branched C ₁ -C ₃₀ alkyl. Exemplary heteroaromatic functional groups include pyridine and derivatives thereof (eg, according to formula (i)), and exemplary aromatic functional groups include benzene and its derivatives (eg according to formula (ii)). And halogen derivatives.

Hyaluronic acid takes the free acid form or the form of a pharmaceutically acceptable salt such as Na, K, Ca, Mg, Zn or Li salt, and the molecular weight is preferably 5 × 10 ^{3 to} 1.6 × 10 ⁶ , more preferably ^{15 × 10 3 ~250 × 10 3} , and most preferably ^{15 × 10 3 ~50 × 10 3} . The preparation method according to the invention consists in dissolving hyaluronic acid in a mixture of water and a water-miscible aprotic solvent, where the solvent is a polar organic solvent and the water content is 10-99. % By volume, preferably 50% by volume. The water miscible aprotic solvent may be, for example, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), acetone, acetonitrile or isopropanol (IPA). The reaction mixture contains R ′ ₃ N base, where R ′ is a linear or branched C _n H _m hydrocarbon chain, where n is an integer from 1 to 4 and m is an integer from 3 to 9. For example, the amount of triethylamine is 0.01 to 20 equivalents, preferably 6 equivalents, relative to the dimer of hyaluronic acid, and the catalyst is selected from the group consisting of substituted pyridines such as dimethylaminopyridine, It is 0.01-1 equivalent with respect to the dimer of hyaluronic acid, Preferably it is 0.05 equivalent. In the preparation process according to the invention, the activation of C ₆ -C ₁₈ -carboxylic acid is first carried out in a polar organic solvent in the presence of a base and 2,4,6-trichlorobenzoic acid or a derivative thereof, or In the presence of chloride, after which a mixture containing activated C ₆ -C ₁₈ -carboxylic acid is added to hyaluronic acid, which is dissolved in a mixture of water, organic solvent, base and catalyst, The product of the reaction obtained is a derivative according to general formula (I). The C ₆ -C ₁₈ -carboxylic acid is selected from the group consisting of caproic acid, enanthic acid, caprylic acid, capric acid, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. The amount of activated C ₆ -C ₁₈ -carboxylic acid is 0.01-5 equivalents, preferably 0.5-2 equivalents, relative to the dimer of hyaluronic acid. Activation of the C ₆ -C ₁₈ -carboxylic acid is carried out at 20 to 60 ° C., preferably 25 ° C., for 5 to 120 minutes, preferably 30 minutes. The reaction between hyaluronic acid and activated C ₆ -C ₁₈ -carboxylic acid is carried out at 20-60 ° C., preferably 25 ° C., for 1-24 hours, preferably 2-3 hours. Thereafter, the C ₆ -C ₁₈ -acylated derivative of hyaluronic acid can be separated from the reaction mixture, washed, dried and lyophilized. The derivative may be separated from the reaction mixture by precipitation using NaCl and alcohol. The derivative may then be washed with alcohol, in particular isopropanol or ethanol.

In a further aspect, the present invention relates to a nanomicelle composition based on a C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to general formula (I), which composition is C-linked to hyaluronic acid. _A nanomicelle comprising a hydrophobic core formed on a _6- C ₁₈ -acyl group and a hydrophilic shell formed on a hydrophilic functional group of hyaluronic acid, wherein one or more biologically active substances are each They are physically bonded in nanomicelles. The composition further includes water and may include a salt (eg, 0.9% NaCl). In a preferred embodiment, the nanomicelle composition comprises 0.3 to 50% by weight of biologically active substance relative to the mass content of the C ₆ -C ₁₈ -acylated derivative of hyaluronic acid, said biologically active substance Is selected from the group consisting of pharmaceutically and cosmetically active substances, in particular vitamins, medicaments, cytostatics, plant extracts, plant complexes or plant active substances, minerals or vegetable oils, or mixtures thereof. Examples of biologically active substances that can be used include tocopherol, paclitaxel, phosphatidylcholine or coenzyme Q10. In a preferred embodiment, the composition comprises a C ₆ -C ₁₈ -acylated derivative of hyaluronic acid at a concentration above its critical aggregation concentration. When the composition is in the form of an aqueous solution, the concentration of the C ₆ -C ₁₈ -acylated derivative of hyaluronic acid is 0.0001 mg · mL ^{−1 to} 30 mg · mL ⁻¹ , preferably ^{1 to} 20 mg · mL ^−1. Range. In another preferred embodiment, the biologically active substance is in an amount of 0.05 to 40% by weight, preferably 1 to 20% by weight, based on the mass content of the C ₆ -C ₁₈ -acylated derivative of hyaluronic acid. Is a mineral or vegetable oil. In yet another preferred embodiment, the composition includes a liquid, water-insoluble biologically active material, the material including an additional biologically active material dissolved therein. Such biologically active substances that are liquid and water-insoluble may be, for example, minerals or vegetable oils, and additional biologically active substances are for example pharmaceutical or cosmetic active substances, in particular vitamins, pharmaceuticals, cytostatics, It may belong to a plant extract, a plant complex or a plant active substance, or a mixture thereof. Nanomicelle compositions according to the present invention may take the form of solutions, nanoemulsions, microemulsions, coacervates or gels.

The invention further relates to a process for the preparation of a nanomicelle composition as defined above, wherein a C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to general formula (I) is dissolved in water. The biologically active substance is dissolved in an organic solvent, the resulting solution is mixed, and then the organic solvent is removed. The organic solvent may be a volatile chlorinated solvent such as trichloromethane, or an alcohol such as ethanol or isopropanol, and the removal may be performed by vacuum evaporation. The aqueous phase is then dried and rehydrated and the resulting nanomicelle structure is filtered and finally lyophilized. Alternatively, the organic solvent may be removed by dialysis. Thereafter, the obtained nanomicelle structure is again filtered and finally freeze-dried. In a preferred embodiment, a C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to general formula (I) is dissolved in water and then mixed with a liquid, water-insoluble biologically active substance, where The resulting mixture is homogenized by sonication to form a microemulsion or nanoemulsion. In another preferred embodiment, a C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to general formula (I) is dissolved in water and then in a liquid in which the additional biologically active substance is dissolved. Mix with the insoluble biologically active material and homogenize the resulting mixture by sonication to form a microemulsion or nanoemulsion.

  In yet another aspect, the present invention relates to the use of nanomicelle compositions for medical or cosmetic applications, preferably topical applications.

式中，ＲはＨ^＋又はＮａ^＋であり，１つ以上のＲ^１メンバーは，不飽和結合を含むことが可能で少なくとも１つの繰り返し単位に存在する直鎖Ｃ_６‐Ｃ_１８鎖，並びに３‐（２‐チエニル）アクリル酸又は３‐（２‐フリル）アクリル酸，又は別の少なくとも１つの繰り返し単位に存在する前記酸の誘導体で表され，そこで，ナノミセル組成物を一般式（II）に従ったＣ_６‐Ｃ_１８‐アシル化ヒアルロナンから調製し，その後，この組成物を架橋反応で安定化する。 Furthermore, a stabilized nanomicelle composition may be prepared. The process for preparing such stabilized nanomicelle compositions consists in preparing C ₆ -C ₁₈ -acylated hyaluronan according to general formula (II):

Wherein R is H ⁺ or Na ⁺ and the one or more R ¹ members can contain unsaturated bonds and are present in at least one repeating unit in a linear C ₆ -C ₁₈ chain, and 3 -(2-thienyl) acrylic acid or 3- (2-furyl) acrylic acid, or a derivative of said acid present in at least one other repeating unit, wherein the nanomicelle composition is represented by the general formula (II) Prepared from the corresponding C ₆ -C ₁₈ -acylated hyaluronan, after which the composition is stabilized by a crosslinking reaction.

ここでは，ヒアルロン酸を水に溶解し，その後，塩基（ＴＥＡなど）及び触媒（ＤＭＡＰ）を溶解し；分離工程では，活性化した３‐（２‐チエニル）アクリル酸又は３‐（２‐フリル）アクリル酸又はそのいずれかの酸の誘導体を調製し，有機溶媒（例えばＴＨＦ）及び塩基（例えばＴＥＡ）に２，４，６‐トリクロロ安息香酸の塩化物を加えた混合液中で活性化を行い，最後に両混合液を混合し，式（III）に従ったアシル化ヒアルロナンを形成する。次いで，一般式（I）に従ったＣ_６‐Ｃ_１８‐アシル化ヒアルロナンの調製方法を参照して上述したものと類似の方法で式（III）に従った前記アシル化ヒアルロナンの活性の為に，活性化Ｃ_６‐Ｃ_１８‐カルボン酸を調製し，式（II）に従ったアシル化ヒアルロナンを形成する。最後に，前記方法で調製したアシル化ヒアルロナンから，ナノミセルを同様に調製することも可能である。このようなナノミセルは，その後に，例えばペルオキシ二硫酸アンモニウムを使用してラジカル反応で架橋してもよい。このような架橋ヒアルロナンは水不溶性である。 In particular, stabilization is carried out by the following method: First, a derivative according to general formula (III) is prepared:

Here, hyaluronic acid is dissolved in water, followed by dissolution of the base (such as TEA) and catalyst (DMAP); in the separation step, activated 3- (2-thienyl) acrylic acid or 3- (2-furyl) ) Prepare a derivative of acrylic acid or any of its acids and activate in a mixture of organic solvent (eg THF) and base (eg TEA) with 2,4,6-trichlorobenzoic acid chloride And finally mix both mixtures to form acylated hyaluronan according to formula (III). For the activity of said acylated hyaluronan according to formula (III) in a manner similar to that described above with reference to the process for preparing C ₆ -C ₁₈ -acylated hyaluronan according to general formula (I) , Activated C ₆ -C ₁₈ -carboxylic acid is prepared to form an acylated hyaluronan according to formula (II). Finally, nano micelles can be similarly prepared from the acylated hyaluronan prepared by the above method. Such nanomicelles may then be crosslinked by a radical reaction, for example using ammonium peroxydisulfate. Such crosslinked hyaluronan is insoluble in water.

The critical micelle (aggregation) concentration (CMC) measurement using the fluorescence method with acylated hyaluronan derivatives (C6) and (C16) containing encapsulated Nile Red in water is shown. The measurement of critical micelle (aggregation) concentration (CMC) using the static light scattering method with acylated hyaluronan derivatives (C6) and (C16) containing encapsulated oil red in water is shown. It is a Cry-SEM image of hyaluronan nano micelles containing encapsulated vitamin E (upper image) and paclitaxel (lower image). Figure 8 shows the cytotoxicity of paclitaxel with concentration and cell interaction time, and the cytotoxicity of paclitaxel encapsulated in HAC6 and HAC16. 1 shows transfer of doxorubicin to HCT116 and MCF-7 cells 1 hour after exposure. 7 shows intracellular translocation of 7AAD in living cells using HA (C6) carrier with encapsulated 7AAD. The profile regarding release of encapsulated oil red from HAC6 carrier to PBS and PBS supplemented with 1% of TWEEN80 is shown. Concentration of HAC6 + oil red: 1 or 10 mg · mL ⁻¹ . FIG. 6 shows the profile of encapsulated paclitaxel released from a HAC6 and HAC16 (c = 10 mg · mL ⁻¹ ) carrier into PBS. The contrast between the penetration of encapsulated Nile Red (NR) from hyaluronan nanomicelles HA (C6) and HA (C10) into the skin and the penetration of Nile Red dissolved in oil and dispersed in water is shown. Each Nile Red sample was at the same concentration. The contrast between penetration of encapsulated Nile Red (NR) from hyaluronan nano micelles HA (C6) and HA (C10) into hair and penetration of Nile Red dissolved in oil and dispersed in water is shown. Each Nile Red sample had the same concentration (the microscopic image shows a cross section of one hair). Comparison of penetration of encapsulated Nile Red (NR) from polymer hyaluronan nanomicelles HA (C6) and HA (C10) into the buccal mucosa and penetration of Nile Red dissolved in oil and dispersed in water into the buccal mucosa Indicates. Each Nile Red sample was at the same concentration. Comparison of penetration of encapsulated Nile Red (NR) from polymer hyaluronan nanomicelles HA (C6) and HA (C10) into the vaginal mucosa and penetration of Nile Red dissolved in oil and dispersed in water Indicates. Each Nile Red sample was at the same concentration.

DS = degree of substitution = 100% of bound substituents ^* molar amount / molar amount of total polysaccharide dimer Unless otherwise specified, the expression “equivalent” (eq) used herein is the dimer of hyaluronic acid. Means for the body. Unless otherwise specified, percentages are calculated on a weight / weight basis.
The molecular weight of most hyaluronic acids was determined by the SEC-MALLS method (source: Contipro Biotech srochenim omezenim, Dornidbroke, Czech).

Example 1 Preparation of capronyl (C6) derivative of hyaluronic acid with mixed aldehyde of 2,4,6-trichlorobenzoic acid and caproic acid 1 g of sodium hyaluronate (2.5 mmol, 15 kDa) in 10 mL of demineralized water Dissolved. Thereafter, 5 mL of DMSO was gradually added. TEA (1.05 mL, 3 eq) and DMAP (8.0 mg, 0.05 eq) were then added to the solution. Simultaneously hexanoic acid (0.63 mL, 2 eq) is dissolved in 5 mL DMSO and TEA (1.05 mL, 3 eq), then 2,4,6-trichlorobenzoyl chloride (1.6 mL, 4 eq) is added to the solution did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with aqueous isopropanol (85% by volume) first to remove DMSO and DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 60% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 4H, γ, δCH ₂ ), δ0.8 (m, 3H, CH ₃ )

Example 2 Preparation of Capronyl (C6) Derivative of Hyaluronic Acid with Mixed Aldehyde of 2,4,6-Trichlorobenzoic Acid and Caproic Acid 1 g of sodium hyaluronate (2.5 mmol, 38 kDa) was dissolved in 10 mL of demineralized water. Thereafter, 5 mL of isopropanol was gradually added. TEA (1.05 mL, 3 eq) and pyridine (0.4 mL, 2.0 eq) were then added to the solution. Simultaneously, hexanoic acid (0.32 mL, 1 eq) is dissolved in 5 mL isopropanol, then TEA (1.05 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.391 mL, 1 eq) are added to the solution did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.50 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with aqueous isopropanol (85% by volume) first to remove pyridine from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS15% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 4H, γ, δCH ₂ ), δ0.8 (m, 3H, CH ₃ )

Example 3 Preparation of Enantyl (C7) Derivative of Hyaluronic Acid with Mixed Aldehyde of 2,4,6-Trichlorobenzoic Acid and Enanthic Acid 1 g of sodium hyaluronate (2.5 mmol, 15 kDa) was dissolved in 10 mL of demineralized water. Thereafter, 5 mL of acetonitrile was gradually added. TEA (0.70 mL, 2 eq) and DMAP (15.0 mg, 0.05 eq) were then added to the solution. At the same time, enanthic acid (0.35 mL, 1 eq) is dissolved in 5 mL of acetonitrile, and then TEA (0.70 mL, 2 eq) and 2,4,6-trichlorobenzoyl chloride (0.39 mL, 1 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.75 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with aqueous isopropanol (85% by volume) first to remove acetonitrile and DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS12% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 6H, γ, δ, ε ( CH ₂ ) ₃ ), δ 0.8 (m, 3H, CH ₃ )

Example 4 Preparation of Caprylyl (C8) Derivative of Hyaluronic Acid with Mixed Aldehyde of 2,4,6-Trichlorobenzoic Acid and Caprylic Acid 1 g of sodium hyaluronate (2.5 mmol, 15 kDa) was dissolved in 10 mL of demineralized water. Thereafter, 5 mL of acetonitrile was gradually added. TEA (1.05 mL, 3 eq) and DMAP (8.0 mg, 0.05 eq) were then added to the solution. At the same time, octanoic acid (0.63 g, 4 eq) is dissolved in 5 mL of acetonitrile, and then TEA (1.05 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.8 mL, 4 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.50 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with aqueous isopropanol (85% by volume) first to remove acetonitrile and DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 40% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 8H, (CH ₂ ) ₄ ) , Δ0.8 (m, 3H, CH ₃ )

Example 5 Preparation of caprinyl (C10) derivative of hyaluronic acid with mixed aldehydes of 2,4,6-trichlorobenzoic acid and capric acid 1 g of sodium hyaluronate (2.5 mmol, 15 kDa) in 10 mL of demineralized water Dissolved. Thereafter, 5 mL of THF was gradually added. TEA (1.05 mL, 3 eq) and DMAP (8.0 mg, 0.025 eq) were then added to the solution. At the same time decanoic acid (0.8 g, 2 eq) is dissolved in 5 mL of THF, then TEA (1.05 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.8 mL, 2 eq) are added to the solution did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS15% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 12H, γ, δ, ε, ζ, η, θCH ₂ ), δ 0.8 (m, 3H, CH ₃ )

Example 6 Preparation of Caprinyl (C10) Derivative of Hyaluronic Acid with Mixed Aldehyde of 2,4,6-Trichlorobenzoic Acid and Capric Acid 1 g of sodium hyaluronate (2.5 mmol, 15 kDa) was dissolved in 10 mL of demineralized water. Thereafter, 5 mL of THF was gradually added. TEA (1.05 mL, 3 eq) and DMAP (8.0 mg, 0.025 eq) were then added to the solution. At the same time, decanoic acid (0.8 g, 4 eq) is dissolved in 5 mL of THF, and then TEA (1.05 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.8 mL, 4 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with aqueous isopropanol (85% by volume) first to remove THF and DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 40% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 12H, γ, δ, ε, ζ, η, θCH ₂ ), δ 0.8 (m, 3H, CH ₃ )

Example 7 Preparation of palmitoyl (C16) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and palmitic acid 0.5 g sodium hyaluronate (1.25 mmol, 38 kDa) dissolved in 20 mL demineralized water did. Thereafter, 10 mL of THF was gradually added. TEA (0.52 mL, 3 eq) and DMAP (8.0 mg, 0.05 eq) were then added to the solution. At the same time, palmitic acid (0.16 g, 0.5 eq) is dissolved in 10 mL of THF, and then TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.098 mL, 0.5 eq) are added. Added to the solution. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 24H, (CH ₂ ) ₁₂ ) , Δ 0.8 (m, 3H, CH ₃ ). DS 14% (measured from NMR)

Example 8 Preparation of stearyl (C18) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and stearic acid 0.5 g sodium hyaluronate (1.25 mmol, 15 kDa) dissolved in 10 mL demineralized water did. Thereafter, 5 mL of THF was gradually added. TEA (0.52 mL, 3 eq) and DMAP (8.0 mg, 0.05 eq) were then added to the solution. At the same time stearic acid (0.711 g, 2 eq) is dissolved in 5 mL of THF, then TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.391 mL, 2 eq) are added to the solution did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. After reacting at room temperature for 3 hours, the reaction mixture was warmed at 50 ° C. for 1 hour. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 7% (measured from NMR)
¹ H NMR (D ₂ O) acyl signal: δ 2.4 ppm (m, 2H, αCH ₂ ), δ 1.6 ppm (m, 2H, βCH ₂ ), δ 1.3 ppm (m, 28H, (CH ₂ ) ₁₄ ) , Δ0.8 (m, 3H, CH ₃ )

Example 9 Preparation of an oleyl (C18: 1) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and oleic acid 0.5 g of sodium hyaluronate (1.25 mmol, 15 kDa) in 10 mL of demineralized water Dissolved in. Thereafter, 5 mL of THF was gradually added. TEA (0.52 mL, 3 eq) and DMAP (15.0 mg, 0.1 eq) were then added to the solution. At the same time, oleic acid (0.18 g, 0.5 eq) is dissolved in 5 mL of THF followed by TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.098 mL, 0.5 eq). Added to the solution. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 10% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 20H, (—CH ₂ —) ₁₀ ),
δ 1.60 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.41 (t, 2H, —CH ₂ —CO—), δ 5.41 (d, 2H, CH = CH)

Example 10 Preparation of an oleyl (C18: 1) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and oleic acid 0.5 g of sodium hyaluronate (1.25 mmol, 130 kDa) in 5 mL of demineralized water Dissolved in. Thereafter, 3 mL of isopropanol was gradually added. TEA (0.52 mL, 3 eq) and DMAP (15.0 mg, 0.1 eq) were then added to the solution. Simultaneously oleic acid (0.4 mL, 1 eq) is dissolved in 5 mL of isopropanol, then TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.195 mL, 1 eq) are added to the solution did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS12% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 20H, (—CH ₂ —) ₁₀ ),
δ 1.60 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.41 (t, 2H, —CH ₂ —CO—), δ 5.41 (d, 2H, CH = CH)

Example 11 Preparation of an oleyl (C18: 1) derivative of hyaluronic acid with a mixed aldehyde of isobutyryl chloride and oleic acid 1.0 g of sodium hyaluronate (2.5 mmol, 15 kDa) was dissolved in 10 mL of demineralized water. Thereafter, 5 mL of THF was gradually added. TEA (1.05 mL, 3 eq) and DMAP (15.0 mg, 0.05 eq) were then added to the solution. At the same time, oleic acid (0.787 mL, 1 eq) was dissolved in 5 mL THF, and then TEA (1.05 mL, 3 eq) and isobutyryl chloride (0.26 mL, 1 eq) were added to the solution. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.50 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 11% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 20H, (—CH ₂ —) ₁₀ ),
δ 1.60 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.41 (t, 2H, —CH ₂ —CO—), δ 5.41 (d, 2H, CH = CH)

Example 12 Preparation of an oleyl (C18: 1) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and oleic acid 0.5 g of sodium hyaluronate (1.25 mmol, 130 kDa) in 5 mL of demineralized water Dissolved in. Thereafter, 3 mL of THF was gradually added. TEA (1.2 mL, 3 eq) and DMAP (15.0 mg, 0.1 eq) were then added to the solution. At the same time oleic acid (0.787 g, 2 eq) is dissolved in 10 mL THF, then TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.391 mL, 2 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS18% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 20H, (—CH ₂ —) ₁₀ ),
δ 1.60 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.41 (t, 2H, —CH ₂ —CO—), δ 5.41 (d, 2H, CH = CH)

Example 13 Preparation of linoleyl (C18: 2) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and linoleic acid 0.5 g of sodium hyaluronate (1.25 mmol, 15 kDa) in 10 mL of demineralized water Dissolved in. TEA (0.52 mL, 3 eq) and DMAP (8 mg, 0.05 eq) were then added to the solution. At the same time, linoleic acid (0.77 mL, 2 eq) is dissolved in 3 mL of THF, and then TEA (1.2 mL, 7 eq) and 2,4,6-trichlorobenzoyl chloride (0.391 mL, 2 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.5 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS16% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 14H, (—CH ₂ —) ₇ ),
δ 1.63 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.44 (t, 2H, —CH ₂ —CO—), _{δ2.83 (m, 2H, = CH} -CH 2 -CH =),
δ 5.45 (m, 4H, CH = CH)

Example 14 Preparation of linoleyl (C18: 2) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and linoleic acid 0.5 g of sodium hyaluronate (1.25 mmol, 15 kDa) in 10 mL of demineralized water Dissolved in. TEA (0.52 mL, 3 eq) and DMAP (8 mg, 0.05 eq) were then added to the solution. At the same time, linoleic acid (0.77 mL, 2 eq) is dissolved in 3 mL of THF, and then TEA (1.2 mL, 7 eq) and 2,4,6-trichlorobenzoyl chloride (0.391 mL, 2 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. After reacting at room temperature for 3 hours, the reaction mixture was warmed at 50 ° C. for 1 hour. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.75 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 20% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 14H, (—CH ₂ —) ₇ ),
δ 1.63 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.44 (t, 2H, —CH ₂ —CO—), _{δ2.83 (m, 2H, = CH} -CH 2 -CH =),
δ 5.45 (m, 4H, CH = CH)

Example 15 Preparation of linolenyl (C18: 3) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and linolenic acid 0.5 g of sodium hyaluronate (1.25 mmol, 15 kDa) in 10 mL of demineralized water Dissolved in. TEA (0.52 mL, 3 eq) and DMAP (8 mg, 0.05 eq) were then added to the solution. At the same time, linolenic acid (0.765 mL, 2.0 eq) is dissolved in 5 mL of THF, and then TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.391 mL, 2.0 eq) are added. Added to the solution. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. The reaction was allowed to proceed for 3 hours at room temperature. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.75 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS15% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 8H, (—CH ₂ —) ₄ ),
δ 1.61 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.43 (t, 2H, —CH ₂ —CO—), δ 2.83 (m, 4H, = CH-CH ₂ -CH =),
δ 5.45 (m, 6H, CH = CH)

Example 16 Preparation of linolenyl (C18: 3) derivative of hyaluronic acid with a mixed aldehyde of 2,4,6-trichlorobenzoic acid and linolenic acid 0.5 g of sodium hyaluronate (1.25 mmol, 15 kDa) in 10 mL of demineralized water Dissolved in. TEA (0.52 mL, 3 eq) and DMAP (8 mg, 0.05 eq) were then added to the solution. At the same time, linolenic acid (0.382 mL, 1 eq) is dissolved in 5 mL of THF, and then TEA (0.52 mL, 3 eq) and 2,4,6-trichlorobenzoyl chloride (0.195 mL, 1 eq) are added to the solution. did. After acid activation, the precipitate was filtered and placed in a prepared solution of HA. After reacting at room temperature for 3 hours, the reaction mixture was warmed at 50 ° C. for 1 hour. The reaction mixture was then diluted with 5 mL of demineralized water to which 0.25 g NaCl was added. In the subsequent precipitation step, 4 times anhydrous isopropanol was used and the acylated derivative was isolated from the reaction mixture. After decantation, the precipitate was washed repeatedly with an aqueous isopropanol solution (85% by volume) first to remove DMAP from the derivative and then with anhydrous isopropanol to remove water from the derivative. The precipitate was then dried at 40 ° C. for 48 hours and then lyophilized to remove residual solvent.
DS 10% (measured from NMR)
¹ H NMR (D ₂ O): δ 0.88 (t, 3H, —CH ₂ —CH ₃ ), δ 1.22-1.35 (m, 8H, (—CH ₂ —) ₄ ),
δ 1.61 (m, 2H, —CH ₂ —CH ₂ —CO—), δ 2.0 ppm (m, 4H, (CH ₂ ) ₂ ), δ 2.43 (t, 2H, —CH ₂ —CO—), δ 2.83 (m, 4H, = CH-CH ₂ -CH =),
δ 5.45 (m, 6H, CH = CH)

Example 17 Encapsulation of Tocopherol (Vitamin E) in Capronyl (C6) Derivative of Hyaluronic Acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 5 mL water with continuous stirring for 3 hours. Tocopherol solution (10 mg in ₃ mL of CHCl ₃ ) was gradually added to the resulting solution with continuous stirring at 25 to 40 ° C., and then 3 mL of CHCl ₃ was gradually added. The CHCl ₃ was then removed from the solution in a continuous evaporation process. After removal of CHCl ₃ , the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
The amount of bound tocopherol (determined by HPLC method) was: 2.3% (w / w).

Example 18 Encapsulation of Nile Red in Capronyl (C6) Derivative of Hyaluronic Acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 5 mL of water with continuous stirring for 3 hours. Nile red solution (10 mg in ₃ mL CHCl ₃ ) was gradually added to the resulting solution with continuous stirring at 25-40 ° C., followed by a further 3 mL CHCl ₃ . The CHCl ₃ was then removed from the solution in a continuous evaporation process. After removal of CHCl ₃ , the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
The amount of Nile Red bound (measured by UV-Vis method) was: 0.4% (w / w).

Example 19 Encapsulation of paclitaxel in capronyl (C6) derivative of hyaluronic acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 5 mL of water with continuous stirring for 3 hours. A paclitaxel solution (10 mg in ₃ mL of CHCl ₃ ) was gradually added to the resulting solution with continuous stirring at 25 to 40 ° C., and then 3 mL of CHCl ₃ was gradually added. The CHCl ₃ was then removed from the solution in a continuous evaporation process. After removal of CHCl ₃ , the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
Bound paclitaxel amount (measured by HPLC method): 5% (w / w)

Example 20 Encapsulation of phosphatidylcholine in capronyl (C6) derivative of hyaluronic acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 5 mL of water with continuous stirring for 3 hours. A phosphatidylcholine solution (10 mg in 5 mL EtOH) was added slowly (dropwise) to the resulting solution with continuous stirring. EtOH was removed from the solution in a continuous evaporation step. The residual aqueous phase was then completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
Amount of bound phosphatidylcholine (measured by HPLC method): 3.0% (w / w)

Example 21 Encapsulation of Coenzyme Q10 in Palmitoyl (C16) Derivative of Hyaluronic Acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 7 was dissolved in 10 mL water with continuous stirring overnight. Coenzyme Q10 solution (20 mg in 5 mL of CHCl ₃ ) was gradually added to the resulting solution with continuous stirring at 30 to 40 ° C., and then 3 mL of CHCl ₃ was gradually added. The CHCl ₃ was then removed from the solution in a continuous evaporation process. After removal of CHCl ₃ , the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
The amount of coenzyme Q10 bound (measured by the UV-Vis method) was: 12% (w / w).
When the product is dissolved in a 0.9% solution of NaCl, a coacervate or gel solution is formed depending on the concentration of the dissolved product.

Example 22 Encapsulation of Tocopherol (Vitamin E) in Stearyl (C18) Derivative of Hyaluronic Acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 8 was dissolved in 10 mL of water with continuous stirring overnight. Tocopherol (about 50 mg in 5 mL of ethanol) was gradually added to the resulting solution with continuous stirring at 25-40 ° C. The ethanol was then removed from the solution in a continuous evaporation process. After removal of EtOH, the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
The amount of bound tocopherol (determined by the UV-Vis method) was 30% (w / w).

Example 23 Encapsulation of tocopherol (vitamin E) in an oleyl (C18: 1) derivative of hyaluronic acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 9 was dissolved in 10 mL of water with continuous stirring overnight. . Tocopherol (about 50 mg in 5 mL of isopropanol) was gradually added to the resulting solution with continuous stirring at 25-40 ° C. The isopropanol was then removed from the solution in a continuous evaporation process. After removal of isopropanol, the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
The amount of bound tocopherol (determined by the UV-Vis method) was: 40% (w / w).

Example 24 Encapsulation of Coenzyme Q10 in Palmitoyl (C16) Derivative of Hyaluronic Acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 7 was dissolved in 10 mL water with continuous stirring overnight. Coenzyme Q10 solution (about 30 mg in 2 mL EtOH) was slowly added to the resulting solution with continuous stirring. After stirring for 3 hours, the resulting mixture was subjected to sonication (100 W) for 30 minutes. The mixture was then dialyzed intensively against distilled water (2 days), filtered through a 1 μm glass filter and lyophilized.
The amount of coenzyme Q10 bound (measured by the UV-Vis method) was: 4.6% (w / w).
When the product is dissolved in a 0.9% solution of NaCl, a coacervate or gel solution is formed depending on the concentration of the dissolved product.

Example 25 Encapsulation of paclitaxel in palmitoyl (C16) derivative of hyaluronic acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 7 was dissolved in 10 mL of water with continuous stirring overnight. To the resulting solution was added paclitaxel solution (about 40 mg in 2 mL EtOH) slowly with continuous stirring. After stirring for 3 hours, the resulting mixture was subjected to sonication (100 W) for 30 minutes. The mixture was then dialyzed intensively against distilled water (3.5 kDa cutoff), filtered through an S4 ceramic frit and lyophilized.
Bound paclitaxel amount (determined by HPLC method): 25% (w / w)

Example 26 Encapsulation of hop extract in an oleyl (C18: 1) derivative of hyaluronic acid 100 mg of an acylated derivative of hyaluronan prepared according to Example 9 was dissolved in 10 mL of water with continuous stirring overnight. Hop extract blend (about 50 mg in 5 mL isopropanol) was slowly added to the resulting solution with continuous stirring. The isopropanol was then removed from the solution in a continuous evaporation process. After removal of isopropanol, the aqueous phase was completely dried, rehydrated in a water bath and filtered through a 1 μm glass filter. The filtrate was lyophilized.
The amount of bound hop extract (measured by UV-Vis method) was: 40% (w / w).

Example 27 Measurement of critical micelle (aggregation) concentration of acylated derivative of hyaluronic acid (a) Fluorescence method The critical micelle (aggregation) concentration was measured from the dependence of fluorescence intensity on the solution concentration (FIG. 1). Nyl red bound acylated derivatives HAC6 (DS = 60%) and HAC16 (DS = 14%), the concentration range of which was adjusted to 0.00002-1.5 mg · mL ⁻¹ according to the procedure described in Example 18 The emission spectrum (580-700 nm) of the aqueous solution was measured with a fluorescence measuring apparatus RF-5301 (Shimadzu) operating at an excitation wavelength of 543 nm.
The following critical micelle (aggregation) concentrations were measured: HAC6: 0.001-0.003 mg · mL ⁻¹ , HAC16: 0.00006-0.0002 mg · mL ⁻¹ (FIG. 1). Similar measurement values were obtained with PBS solution containing 0.9% NaCl.
(B) Static Light Scattering Method The critical micelle (aggregation) concentration was measured from the dependence of the scattered light intensity (I ₉₀ ) on the solution concentration (FIG. 2). Nyl red bound acylated derivatives HAC6 (DS = 60%) and HAC16 (DS = 14%) prepared in a concentration range of 0.00002 to 0.06 mg · mL ⁻¹ according to the procedure described in Example 18 The scattered light intensity in the aqueous solution was measured at an angle of 90 ° with a photometer DAWN EOS (Wyatt Technology Corporation) having an operating wavelength of 632 nm.
The following critical micelle (aggregation) concentrations were measured: HAC6: 0.002-0.004 mg · mL ⁻¹ , HAC16: 0.00006-0.0001 mg · mL ⁻¹ (FIG. 2). Similar measurement values were obtained with PBS solution containing 0.9% NaCl.

Example 28 Measurement of zeta potential of hyaluronan nano micelles The zeta potential was measured with an apparatus Zetasizer Nano-ZS (Malvern Instruments) equipped with a He-Ne laser (633 nm). Regardless of the encapsulated material, the zeta potential exhibited by the nanomicelle in the aqueous solution was ˜-50 mV at 5 mg · mL ⁻¹ and ˜60 to −70 mV after 10-fold dilution. In 0.9% NaCl solution, the zeta potential range decreased (-30 to -23 mV). Therefore, the absolute value of the zeta potential shows a high stability of the prepared nanomicelle in an aqueous solution and a relatively high stability in a salt solution.

Example 29 Morphological Analysis of Hyaluronan Nanomicelles Microscopic analysis was performed at −135 ° C. in a scanning microscope JEOL7401F with a beam acceleration voltage of 2 kV (ie fine beam mode). In order to perform the above analysis, 2-3 μL of a concentrated sample (about 20 mg / 0.4 mL) was dropped onto an Al plate and immersed in liquid nitrogen filled in a cooling chamber Alta2500 (Gatan). They were then coated with a Pt / Pd mixture for 2 minutes.
Nanomicelle dimensions of hyaluronan C6 with encapsulated vitamin E (Example 17) and hyaluronan C16 with encapsulated paclitaxel (Example 25) ranged from 20 to 50 nm (Figure 3).

表１に収載された結果から，ヒアルロナンナノミセル中のアシル化鎖の分布は主にアシル鎖の長さに影響されることが明らかに分かる。アシル鎖の凝集度はその長さの増加に伴って増加する。ナノミセルへの非極性物質の取り込みは，個々のアシル鎖の長さに無関係に起こる。この特定のケースでは，ミセル中の非極性物質の分布は常に完全に行き渡っている（＞９９．５%）。 Example 30 Distribution of acylated chains and non-polar substances in hyaluronan nanomicelles Hydrophobized hyaluronan nanomicelles containing encapsulated vitamin E were separated by flow field flow fractionation (FlFFF) method using frit inlet separation channel. . For analysis purposes, 10 mg of lyophilized acylated hyaluronan conjugated with vitamin E (prepared from the derivatives listed in Table 1 according to the procedure described in Example 23) was added to 1 mL of mobile phase (50 mM NaNO ₃ and 0.02% Of NaN ₃ ) and filtered through a glass syringe filter with a pore size of 1 μm. Thereafter, 100 μl was injected into the FlFFF apparatus.
Separations were performed with a cross-flow gradient of 2 mL / min to 0.1 mL / min at 5 min intervals. The set value of the flow rate of the mobile phase supplied to the detector was kept constant at 1 mL / min.
Separation took place at the experimental temperature. The eluate was observed with a light scattering detector DAWN EOS, a differential refractometer Optilab rEX (both manufactured by Wyatt Technology Corporation) and a UV detector (Shimadzu) with an operating wavelength of 292 nm.
When the above method is applied, it is possible to measure both the proportion of the binding substance and the hydrophobized hyaluronan that are reliably incorporated inside the nano micelles and the proportion of those existing outside the aggregated structure (Table 1). reference).
Table 1. Distribution of acylated chains and vitamin E (tocopherol) inside and outside hyaluronan aggregates (nano micelles)

From the results listed in Table 1, it is clear that the distribution of acylated chains in hyaluronan nanomicelles is mainly influenced by the length of the acyl chains. The degree of acyl chain aggregation increases with increasing length. Incorporation of nonpolar substances into nanomicelles occurs regardless of the length of the individual acyl chains. In this particular case, the distribution of nonpolar substances in the micelles is always fully distributed (> 99.5%).

Example 31 Cytotoxicity of Nanomicelles Carrying Paclitaxel-Based Cell Growth Inhibitors Paclitaxel conjugated to hyaluronan C6 and C16 acylated derivatives prepared according to the procedures described in Examples 19 and 25, respectively, was added to the culture medium (10 % Final FBS) to a final concentration of 100 μg / mL. To test paclitaxel at concentrations of 0.001, 0.01, 0.1, 1.0, 10.0 and 100.0 μg / mL supported on acylated hyaluronan C6 and C16 derivatives, Cells (NHDF) cells, human breast cancer cell line (MCF-7) and human colon cancer cell line (HCT116) were used and tested based on cell viability measurements. The effect of paclitaxel supported on acylated hyaluronan C6 and C16 derivatives was compared to the effect of paclitaxel on the hyaluronan itself (FIG. 4). Cell viability measurements are based on the detection of dehydrogenase activity, which becomes active in living cells and turns yellow substances into purple solutions. The absorbance of the enzyme detected at 540 nm is proportional to the proportion of living cells.
In particular, when HAC16 was used, the cell growth inhibitory effect of paclitaxel slightly decreased when the carrier concentration was increased (FIG. 4).
The acylated derivative itself did not show any cell growth inhibitory effect.

Example 32 Transfer of Encapsulated Substances to Cells The substances doxorubicin and 7-aminoactinomycin D (7-AAD, which is a substance that only penetrates dead and permeabilized cells), according to the procedure described in Example 18 ( This was encapsulated in an acylated derivative of hyaluronan C6 (where 7-AAD was substituted for Nile Red). To test whether there is a difference in the penetration of a substance into a cell when the substance is added to a solution, when the substance is present by itself and when it is present in an acylated derivative of hyaluronan C6 Human skin fibroblast (NHDF) cells, human breast cancer cell line (MCF-7) and human colon cancer cell line (HCT116) were used. The test was performed by fluorescence microscopy (inverted microscope Nikon Eclipse Ti). Doxorubicin was tested at a concentration of 5.0 μg / mL (FIG. 5) and 7-AAD was tested at a concentration of 15 μg / mL (FIG. 6).
The substance transfer from the carrier to the cells was good.

Example 33 Release of Encapsulated Oil Red from Nanomicelles into Solution Oil Red (Oil Red O, Solvent Red) encapsulated in HAC6 according to the procedure described in Example 18 (where Nile Red was replaced with Oil Red O) The release of 27) into the solvent was examined in vitro. The target solution used was PBS and PBS supplemented with 1% TWEEN80. An aqueous solution (concentration 1 to 10 mg · mL ⁻¹ ) of an acylated derivative bound to oil red is dissolved in PBS or PBS supplemented with 1% TWEEN 80, and quantitatively added to a dialysis tube (MWCO 12 to 14 kDa, Spectrum Laboratories). Transferred and dialyzed at 37 ° C. against PBS or PBS supplemented with 1% TWEEN80. At predetermined time intervals, 4 mL of dialysate was collected and replaced with fresh medium. The amount of oil red released was measured by the UV-Vis method (FIG. 7).
The slow release of the bound substance is an indication that the carrier system is highly stable in PBS.

Example 34 Release of Encapsulated Paclitaxel from Nanomicelles into Solution Release of paclitaxel encapsulated in HAC6 and HAC16 into solvent was studied in vitro at a temperature of 37 ° C. according to the procedures described in Examples 19 and 25, respectively. An acylated derivative aqueous solution containing paclitaxel having a total concentration of 0.2 mg was dissolved in PBS, quantitatively transferred to a dialysis tube (MWCO 12-14 kDa, Spectrum Laboratories), and dialyzed against 50 mL of PBS at a temperature of 37 ° C. The dialysate was replaced with fresh medium at predetermined time intervals. The amount of paclitaxel released was measured by the HPLC method after it was extracted into chloroform, evaporated and then dissolved in acetonitrile (FIG. 8).
The release recorded with HAC16 was slow compared to HAC6. The slow release of the bound substance is an indication that the carrier system is highly stable in PBS.

Example 35 Preparation of nanoemulsions and microemulsions from oleyl derivatives of hyaluronic acid and oleic acid (C18: 1) 80 mg of acylated derivative of hyaluronan prepared according to Example 11 was stirred into 4 mL of water with continuous overnight stirring. Dissolved. To the resulting solution, 8 mg oleic acid was gradually added with continuous stirring. After stirring, the resulting mixture was subjected to two-stage sonication (Ultrasonic Processor, UPS 200S, 200 W output). The first stage is a continuous process (50% amplitude), followed by the second stage, which is a pulsating process (0.8 second pulse, 70% amplitude), each stage lasting 25 minutes. It was. In the sonication process, the container containing the mixed solution was immersed in an ice bath to protect it from overheating.
Particle size (measured with Zetasizer): 200-300 nm. The dimensions depended on the amount of oil phase in the mixture.

Example 36 Preparation of nanoemulsion and microemulsion from hyaluronic acid and oleyl derivative of oleic acid (C18: 1) in which coenzyme Q10 was dissolved 80 mg of acylated derivative of hyaluronan prepared according to example 11 was continuously stirred overnight. While dissolved in 4 mL of water. To the resulting solution, 8 mg of oleic acid in which Coenzyme Q10 (about 0.5 mg) was previously dissolved was continuously added with continuous stirring. After stirring, the resulting mixture was subjected to two-stage sonication (Ultrasonic Processor, UPS 200S, 200 W output). The first stage is a continuous process (50% amplitude), followed by the second stage, which is a pulsating process (0.8 second pulse, 70% amplitude), each stage lasting 25 minutes. It was. In the sonication process, the container containing the mixed solution was immersed in an ice bath to protect it from overheating.
Particle size (measured with Zetasizer): 200-300 nm. The dimensions depended on the amount of oil phase in the mixture.

Example 37 Preparation of stabilized nanomicelles via covalent crosslinking 10 g of sodium hyaluronate (25 mmol, 38 kDa) was dissolved in 200 mL of demineralized water. TEA (6.97 mL, 2 eq for the dimer of HA) and DMAP (153 mg, 0.05 eq) were then added to the solution. Activation: 3.5 g 3- (2-furyl) acrylic acid (25 mmol) was dissolved in 50 mL tetrahydrofuran and 19.2 mL TEA (2 eq). The resulting solution was then cooled in an ice bath and trichlorobenzoyl chloride (1.2 mL) was added. The reaction was allowed for 15 minutes. Thereafter, the activated acid was added to the hyaluronan solution and reacted at 25 ° C. for 24 hours. The resulting reaction mixture was diluted with water. The acrylated derivative was isolated from the reaction mixture in the next precipitation step using 4X anhydrous isopropanol. After decantation, the precipitate was repeatedly washed with an isopropanol aqueous solution (85% by volume). The precipitate was then dried at 40 ° C. for 48 hours. The resulting acylated derivative was acylated (see Example 1), then dissolved in water and lyophilized to remove residual solvent.
According to Example 18 (here, oil red was substituted for Nile Red), a carrier system prepared from Oil Red (Oil red O) and an acylated derivative was dissolved to form a 1% aqueous solution. In the resulting solution, ammonium peroxydisulfate (10 eq) was used as a trigger to crosslink the support system.
DS20% active group for photocrosslinking (measured from NMR)
¹ H NMR (D ₂ O): δ 7.83, 6.78 (d, J = 3.5), 6.78, 6.61 (bs), 7.83, 7.59 (J _trans = 16. 01), 6.39 (J _trans = 15.85)

Example 38 Topical application of hyaluronan nano micelles-penetration into skin, hair and mucosa Porcine skin, bovine nitrogen mucosa and bovine buccal mucosa were donated by MasoEko sro, based in Letohrad, Kuncice 243. Immediately after acquisition, the sample was subjected to the passive action of an acylated hyaluronan C6 and C10 (10 mg · mL ⁻¹ ) solution containing encapsulated Nile Red (prepared according to the procedure described in Example 18), and the inverted microscope Nikon Eclipse The penetration of the sample was compared by measuring the fluorescence value with Ti (with the objective lens Plan Fluor4x). Thereafter, incubation was performed at 37 ° C .: a skin sample for 20 hours (FIG. 9), a hair sample for 15 minutes (FIG. 10), and a mucosa sample for 4 hours (FIGS. 11 and 12).
When compared using an oily solvent and an aqueous solvent, the carrier was more efficient in penetrating the skin, hair and mucous membranes.

Claims (35)

C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to general formula (I):

In the formula, R is H ⁺ or Na ⁺ , R ¹ is H or C (═O) C _x H _y or C (═O) CH═CH-het, and x is an integer of 5 to 17. , Y is an integer from 11 to 35, C _x H _y is a linear or branched saturated or unsaturated C ₅ -C ₁₇ chain, and het can select its content from N, S or O atoms A heterocyclic or heteroaromatic group, wherein at least one repeating unit comprises one or more R ¹ , —C (═O) C _x H _y or C (═O) CH═CH—het groups, and n is Derivatives intended to be in the range of 12-4000. 2. The method according to claim 1, wherein R is H ⁺ or Na ⁺ , and R ¹ is —C (═O) (CH ₂ ) ₇ —CH═CH— (CH ₂ ) ₇ —CH ₃ . C ₆ -C ₁₈ -acylated derivative of hyaluronic acid. In the presence of a base and a catalyst in a mixture of water and a water-miscible aprotic solvent, hyaluronic acid is 2,4,6-trichlorobenzoic acid chloride or R ₃ -CO-Cl: ₃ is an aliphatic or branched C ₁ -C ₃₀ -alkyl, heteroaromatic or aromatic functional group: characterized by reacting with an organic chloride activated C ₆ -C ₁₈ -carboxylic acid A method for preparing a hyaluronic acid derivative according to general formula (I) Hyaluronic acid is in the form of free acid or pharmaceutically acceptable salt, and its molecular weight is preferably 5 × 10 ^{3 to} 1.6 × 10 ⁶ , more preferably 15 × 10 ³ to 250 × 10 ³ , The preparation method according to claim 3, wherein the preparation method is most preferably 15 × 10 ³ to 50 × 10 ³ .   Hyaluronic acid is dissolved in a mixture of water and a water-miscible aprotic solvent, and the solvent is a polar organic solvent, and the water content is 10 to 99% by volume, preferably 50% by volume. The preparation method according to claim 3 or 4.   The preparation method according to any one of claims 3 to 5, wherein the water-miscible aprotic solvent is dimethyl sulfoxide, tetrahydrofuran, acetone, acetonitrile or isopropanol. The reaction mixture is R ′ ₃ N base: where R ′ is a linear or branched C _n H _m hydrocarbon chain, n is an integer from 1 to 4, and m is an integer from 3 to 9. For example, triethylamine is contained in an amount of 0.01 to 20 equivalents, preferably 6 equivalents, relative to the dimer of hyaluronic acid, and the catalyst is selected from a group consisting of substituted pyridines such as dimethylaminopyridine, the amount of which is The preparation method according to any one of claims 3 to 6, wherein the amount is 0.01 to 1 equivalent, preferably 0.05 equivalent, relative to the dimer. First, activation of C ₆ -C ₁₈ -carboxylic acid is carried out in a polar organic solvent in the presence of a base and 2,4,6-trichlorobenzoic acid or a derivative thereof, or in the presence of a base and an organic chloride, Next, a mixed solution containing activated C ₆ -C ₁₈ -carboxylic acid is added to hyaluronic acid, and this is dissolved in a mixed solution of water, an organic solvent, a base, and a catalyst. The preparation method according to any one of claims 3 to 7, which is a derivative according to (I). The C ₆ -C ₁₈ -carboxylic acid is selected from the group consisting of caproic acid, enanthic acid, caprylic acid, capric acid, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. The preparation method of any one of 3-8. The amount of activated C ₆ -C ₁₈ -carboxylic acid is 0.01-5 equivalents, preferably 0.5-2 equivalents, relative to the dimer of hyaluronic acid. The preparation method of any one of -9. Preparation according to any one of claims 3 to 10, characterized in that the activation of the C ₆ -C ₁₈ -carboxylic acid is carried out at 20 to 60 ° C, preferably at 25 ° C, for 5 to 120 minutes, preferably 30 minutes. Method. The reaction of the activated C ₆ -C ₁₈ -carboxylic acid with hyaluronic acid is carried out at 20-60 ° C, preferably 25 ° C, for 1-24 hours, preferably 2-3 hours. The preparation method of any one of 3-11. The C ₆ -C ₁₈ -acylated derivative of hyaluronic acid is subsequently separated from the reaction mixture, washed, dried and lyophilized. 13. Preparation method. Preparation process of claim 13, wherein the separating from the acylated derivatives in the precipitation process using NaCl and alcohol reaction mixture _- C ₆ -C 18 of the hyaluronic acid. Process for the preparation of claim 13 or 14, wherein washing the acylated derivatives alcohols, in particular isopropanol or ethanol _- C ₆ -C 18 of the hyaluronic acid. A nanomicelle composition based on a C ₆ -C ₁₈ -acylated derivative of said hyaluronic acid according to general formula (I), the composition formed on a C ₆ -C ₁₈ -acyl group bound to hyaluronic acid A nanomicelle comprising a hydrophilic shell formed by a hydrophobic core and a hydrophilic functional group of hyaluronic acid, wherein one or more biologically active substances are physically bound in the nanomicelle Nano micelle composition. Containing 0.3 to 50% by weight of a biologically active substance based on the mass content of the C ₆ -C ₁₈ -acylated derivative of hyaluronic acid, the biologically active substance being a pharmaceutically and cosmetically active substance 18. A nanomicelle composition according to claim 16, characterized in that it is selected from the group consisting of vitamins, medicaments, cytostatics, plant extracts, plant complexes or plant active substances, minerals or vegetable oils, or mixtures thereof. .   18. The nanomicelle composition according to claim 17, wherein the biologically active substance is tocopherol, paclitaxel, phosphatidylcholine or coenzyme Q10. As C ₆ -C said hyaluronan concentrations above the critical aggregation concentration 18 _- claim 16 to 18 nano-micelles composition according to any one which comprises an acylated derivative. When the composition is in the form of an aqueous solution, the concentration of the C ₆ -C ₁₈ -acylated derivative of hyaluronic acid is 0.0001 mg · mL ^{−1 to} 30 mg · mL ⁻¹ , preferably ^{1 to} 20 mg · mL ^−1. The nanomicelle composition according to any one of claims 16 to 19, wherein the composition is in the range of. The biologically active substance is a mineral or vegetable oil in an amount of 0.05 to 40% by weight, preferably 1 to 20% by weight, based on the mass content of the C ₆ -C ₁₈ -acylated derivative of the hyaluronic acid. 21. The nanomicelle composition according to any one of claims 16 to 20, wherein the composition is present.   22. A liquid, water-insoluble biologically active substance, wherein said substance contains an additional biologically active substance dissolved therein. The described nano micelle composition.   The liquid and water-insoluble biologically active substance is mineral or vegetable oil, and the additional biologically active substance is a pharmacological or cosmetically active substance, especially vitamins, pharmaceuticals, cytostatics, plant extracts, plant complexes 23. The nanomicelle composition according to claim 22, which belongs to a plant active substance, or a mixture thereof.   24. A nanomicelle composition according to any one of claims 16 to 23, which is in the form of a solution, nanoemulsion, microemulsion, coacervate or gel. The C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to the general formula (I) is dissolved in water, the biologically active substance is dissolved in an organic solvent, and the resulting solution is mixed; 25. A method for preparing a nanomicelle composition as defined in any one of claims 16 to 24, wherein the organic solvent is removed.   26. Preparation according to claim 25, characterized in that the organic solvent is removed by vacuum evaporation, after which the aqueous phase is dried and rehydrated, the resulting nanomicelle structure is filtered and finally freeze-dried. Method.   25. The preparation method according to claim 24, wherein the organic solvent is removed by dialysis, and then the obtained nanomicelle structure is filtered and finally freeze-dried.   The preparation method according to any one of claims 25 to 27, wherein the organic solvent is a volatile chlorinated solvent such as trichloromethane or an alcohol such as ethanol or isopropanol. The C ₆ -C ₁₈ -acylated derivative of hyaluronic acid according to the general formula (I) is dissolved in water and then mixed with a liquid, water-insoluble biologically active substance, and the resulting mixture is A method for preparing a nanomicelle composition as defined in claim 16, characterized in that it is homogenized by sonication to form a microemulsion or nanoemulsion. A C ₆ -C ₁₈ -acylated derivative of the hyaluronic acid according to the general formula (I) is dissolved in water and then a liquid insoluble biological substance in which the additional biologically active substance is dissolved 24. A method for preparing a nanomicelle composition as defined in claim 22 or 23, characterized in that it is mixed with an active substance and the resulting mixture is homogenized by sonication to form a microemulsion or nanoemulsion.   Use of a nanomicelle composition as defined in any one of claims 16 to 24 for medical or cosmetic use, preferably topical application. A process for preparing a stabilized nanomicelle composition comprising preparing a C ₆ -C ₁₈ -acylated hyaluronan according to general formula (II) comprising:

Wherein R is H ⁺ or Na ⁺ , and the one or more R ¹ members can contain unsaturated bonds and are present in at least one repeating unit in a straight chain C ₆ -C ₁₈ chain, and 3 -(2-thienyl) acrylic acid or 3- (2-furyl) acrylic acid, or a derivative of said acid present in at least one other repeating unit, wherein the nanomicelle composition is represented by the general formula (II) A method of preparation in which the composition is prepared from the C ₆ -C ₁₈ -acylated hyaluronan according to claim 1, and then the composition is stabilized by a crosslinking reaction. First, hyaluronic acid with 3- (2-thienyl) acrylic acid or 3- (2-furyl) acrylic acid or a derivative of each acid in water and a water-miscible nonpolar solvent in the presence of a base and a catalyst. Reacting the acid with 2,4,6-trichlorobenzoic acid chloride, or R ₃ —CO—Cl, wherein R ₃ is aliphatic or branched C ₁ -C ₃₀ -alkyl, optionally complex Activated with an organic chloride of: containing an aromatic or aromatic functional group to form an acrylated hyaluronan according to formula (III) above:

The acrylated hyaluronan according to formula (III) is then converted to 2,4,6-trichlorobenzoic acid chloride or R ₃ —CO in water and a water miscible nonpolar solvent in the presence of a base and a catalyst. -Cl: wherein R ₃ is aliphatic or branched C ₁ -C ₃₀ -alkyl, optionally containing heteroaromatic or aromatic functional groups: C ₆ -C _18- activated by organic chlorides of Reacting with a carboxylic acid to form a C ₆ -C ₁₈ -acylated hyaluronan according to formula (II), and then using the method defined in any one of claims 25 to 30 to formula (II) 33. The preparation method according to claim 32, wherein a nano micelle composition is prepared from the hyaluronan according to claim 3 and then subjected to a crosslinking reaction by radical reaction.   The preparation method according to claim 32 or 33, wherein the radical reaction is catalyzed by a crosslinking agent.   The preparation method according to claim 34, wherein the cross-linking agent is ammonium peroxydisulfate.

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